Creation of cross-linkages between polysaccharides of the yeast cell wall by transglycosylation. Construction of the cell wall, a structure external to the plasma membrane, is fraught with problems for the fungal cell: how to synthesize the cell wall material, how to get it across the plasma membrane and how to organize it into an ordered and strong structure that will protect the cell from internal turgor pressure and external stresses. As we and others showed, the components of the cell wall are cross-linked to form a tight network that endows the wall with the strength necessary to perform its functions. The formation of these cross-links poses a thermodynamic puzzle: the components of the cell wall are either made inside the cell and excreted, as for mannoproteins, or synthesized and extruded at the plasma membrane, as in the case of chitin and glucans. They only meet in the periplasmic space, where the usual sources of energy, such as ATP, are missing. So, where does the free energy come for the formation of the new cross-links? Twenty years ago, we tried to answer this question, proposing that the cross-links would be generated by transglycosylation. In this way, the energy of previously made linkages would be used to make new ones: for every bond lost in the process of glycosyl transfer, a new one would be created. Insufficient knowledge about the cell wall structure and lack of tools for the study of cross-link distribution delayed verification of this hypothesis until recently. We determined most of the linkages among cell wall components in 1995-97. During a sabbatical in Salamanca, Spain, I developed a new procedure to measure quantitatively the percentage of chitin in the cell wall that is free, bound to beta(1-3)glucan or bound to beta(1-6)glucan. Using this procedure, and in collaboration with Javier Arroyo of the University of Madrid, Spain, we were able to show that two putative transglycosylases, Crh1p and Crh2p are redundantly required for the attachment of chitin to beta(1-6)glucan, thus supporting the transglycosylation hypothesis.[unreadable] Our further goal was to demonstrate the reaction in vitro, a task that was initiated in the previous period. At this stage, I also had the collaboration of Vladimir Farkas, of the Slovak Academy of Sciences in Bratislava, Slovakia and of Peter McPhie in the Laboratory of Biochemistry and Genetics. Arroyo and coworkers constructed some strains, whereas Farkas and coworkers synthesized most of the fluorescent oligosaccharides used in this study. The bulk of the experiments were carried out in my laboratory. Chitin could not be used directly as a substrate, because of its insolubility, therefore we had to use other means to demonstrate the reaction between this polysaccharide and beta(1-6)glucan. As communicated in last years Report, sulforhodamine-linked oligosaccharides (SR-oligosaccharides) were used as artificial acceptors in the reaction and their incorporation was monitored by observation in the fluorescence microscope. This manner of visualization provided information about the localization of the reaction. When the SR-oligosaccharides were added to growing cells, the cells became fluorescent at the cortex, especially at bud scar sites, which mark previous divisions. As an approach to an in vitro system, digitonin-permeabilized cells were incubated with the SR-oligosaccharides. Here, the addition of UDP-N-acetylglucosamine, the substrate for chitin synthases, was necessary for the uptake of fluorescence, showing that chitin was a partner in the reaction. Moreover, both with intact and permeabilized cells, chitinase digestion solubilized the fluorescence. Mutants lacking both Crh1p and Crh2p did not incorporate fluorescent oligosaccharides. [unreadable] In the present period, our efforts went towards quantification of the results and development of a more artificial system. We found that permeabilized cells of LC355, a strain lacking five beta(1-3)glucanases, after previous growth at 38C and permeabilization, showed extremely high fluorescence after incubation with the SR-oligosaccharides. After release with chitinase, we were able to measure the fluorescence in the spectrofluorimeter and to show that the reaction continued almost linearly for several hours. Furthermore, we had previously shown with growing cells that a gas1 mutant, defective in a protein that remodels beta(1-3)glucan, shows a greatly increased uptake of fluorescence both in growing and permeabilized cells. In the latter, the fluorescence in bud scars was not dependent on the presence of UDP-N-acetylglucosamine, suggesting that the cells were using endogenous chitin for the reaction. Isolated cell walls from gas1 cells showed the same behavior, incorporating fluorescence in bud scars in the absence of UDP-N-acetylglucosamine, thus providing a cell-free system that catalyzes the reaction. Here too, we could quantify the incorporation as a function of time and show that chitinase treatment solubilizes the fluorescence. All these results establish that the linkage between chitin and beta(1-6)glucan takes place by a transfer of part of a chitin chain to a glucan chain and demonstrate for the first time that a linkage between two different polysaccharides can occur by transglycosylation. A paper incorporating these results was recently accepted by the Journal of Biological Chemistry.[unreadable] The effect of hyperpolarized growth on the yeast cell wall. A project, initiated when Martin Schmidt was a postdoctoral fellow in my laboratory, was completed, mostly in his laboratory at Des Moines University. In this study, we showed that cak1 and cla4, two protein kinase mutants that we isolated in a screen for synthetic lethality with chs3 (chitin synthase 3 mutant), show hyperpolarized growth together with thinner and weaker lateral walls. In these mutants, inhibition or elimination of Chs3p activity leads to lysis. These results highlight the importance of chitin for the integrity of the cell wall in a stress situation. A paper on this findings was published in FEMS Yeast Research.